Abstract:

The present invention relates to a three dimensional, malleable cell
culture composition and method of forming the same comprising hyaluronic
acid, chitosan and a polyelectrolytic complex of hyaluronic acid and
chitosan. These three components in combination constitute an initial
microenvironment for support of stromal cells, and their undifferentiated
mesenchymal cell progeny. The tissue engineering device and method of
forming the same comprising hyaluronic acid and chitosan and the use of
said device with compositions of pluripotent cells and various
formulations of cell culture media for repair of tissues is disclosed.

37. The composition of claim 34, wherein the biologically active agent is
paclitaxel.

38. The composition of claim 36, wherein the protein is a member of the
transforming growth factor-beta protein family.

39. The composition of claim 35, wherein said one or more biologically
active agents are present in a portion of said fluid mass composition
selected from the unreacted protonated chitosan hydrogel, the unreacted
hyaluronic acid viscoelastic gel and a combination thereof.

Description:

FIELD

[0001]The present invention relates generally to malleable cell culture
composition and methods of forming the same. The composition preferably
comprises hyaluronic acid, chitosan and a polyelectrolytic complex
comprised of hyaluronic acid and chitosan. It is believed that these
components in combination constitute an initial microenvironment for
support of stromal cells, and their undifferentiated mesenchymal cell
progeny.

BACKGROUND

[0002]The field of tissue engineering relates to the technology of
generating molecules, cells, tissues and, in rare instances, complete
organs suitable for regenerating phenotypically specific tissue in host
defects or injuries. Fundamental to the success of these efforts is
understanding the mechanisms of cellular tensegrity by which
undifferentiated pluripotent mesenchymal cells interpret information they
receive from their microenvironments and translate these signals into
biochemical messages capable of influencing expression of the genome.

[0003]Traditional in vitro cell culture methods employ two dimensional,
polystyrene cell culture plates. While much valuable information has been
discovered in such systems, two significant issues limit their
application to tissue engineering problems. First, in vitro culture
plates are generally rigid structures made, typically, of polystyrene.
Secondly, in vitro culture plates provide the cells with only a two
dimensional substratum. This two dimensional substratum is restrictive,
since cells of tissues and organs respond to signals initiated within a
three dimensional (3-D) microenvironment. Cells comprising all tissues,
including bone, have some capacity to alter their three dimensional
morphologies in response to changes in mechanical forces present within
their environments.

[0004]Tissue engineering research has focused on the regeneration of bone
and articular cartilage. Some remarkable successes toward these ends have
been achieved by employing bioresorbable, synthetic compounds to
fabricate anatomically and/or functionally specific, 3-D architectures by
which biologically active agents and/or living cells are presented to
bone or cartilage defects. One approach involves isolating cells from the
body, expanding them in in vitro cultures, placing them on or within
structural matrices, and implanting the new system inside the body. A
commercial example of this approach is Carticell® (Genzyme Corp.,
Boston, Mass.), wherein a process and device that expands a patient's own
articular cartilage cells (chondrocytes) in vitro and return them within
a type I collagen matrix for treatment of articular cartilage defects.
Another commercial construct known as Infuse®
(Medtronic-Softmore-Danek, Memphis, Tenn.) is a device composed of a type
I collagen sponge saturated with a protein known as bone morphogenetic
protein (recombinant human bone morphogenetic protein-2 [rh-BMP-2]). This
saturated sponge induces bone formation within the interbody spinal
fusion model in the human.

[0005]Tissue engineering research has been directed toward: identification
of appropriate cell sources (mature, pluripotent progenitor and stem
cells); selection of suitable bioactive molecules (growth factors and
morphogens); and fabricating devices of various compositions and
geometries (synthetic or natural polymers, meshes or foams) to function
as cell culture substrata. A three dimensional substratum may be
described as an intricate biocompatible and bioresorbable network of
natural or synthetic fibers defining an internal organization of spaces
(voids) within which cells can grow, migrate, proliferate and
differentiate if they are provided with a suitable extracellular matrix
(ECM), and appropriately formulated nutrient media either in vitro or in
vivo.

[0007]Mesenchymal stem cells (MSC) are the formative pluripotential blast
cells found inter alia in association with capillaries (i.e., the
vascular pericyte) and bone marrow that are capable of differentiating
into any of the specific types of connective tissue cells such as
adipocytes, osteoblasts, chondrocytes, fibroblasts and myocytes (of
smooth and skeletal muscle as well as the cardiomyocyte). Phenotype
selection for the MSC is directed by various influences exerted by
soluble bioactive factors such growth factors, morphogens and cytokines
as well as information derived from their microenvironments by means of
mechanochemical signal transduction. Although these cells are normally
present at very low frequencies in bone marrow, through a process
disclosed by Caplan and Haynesworth in U.S. Pat. No. 5,486,359 these
cells may be isolated, purified and replicated in culture.

[0008]In order to isolate human mesenchymal stem cells (h-MSC), it is
necessary to isolate rare pluripotent mesenchymal stem cells from other
cells in the bone marrow or other MSC sources. Bone marrow cells may be
obtained from the iliac crest, femur, tibia, spine, rib or other
medullary spaces. Other sources of human mesenchymal stem cells include
embryonic yolk sac, placenta, umbilical cord, fetal and adolescent skin,
blood and other mesenchymal stem cell tissues.

[0009]Isolated human mesenchymal stem cell (h-MSC) compositions serve as
progenitors for various mesenchymal cell lineages. Isolated mesenchymal
stem cell populations have the ability to expand in culture without
differentiating and have the ability to differentiate along specific
connective tissue lineages when cultured in vitro or introduced in vivo
at a place of damaged tissue. In order to realize the therapeutic
potential of these cells to restore diseased or damaged connective
tissues, they must be entrusted to a delivery vehicle possessed of
biochemical and mechanical properties appropriate for propagation of a
particular cell phenotype.

[0010]Several patents have attempted to describe a suitable biocompatible
delivery vehicle for both undifferentiated and phenotypically mature
cells. For example, U.S. Pat. No. 6,482,231 discloses a biological
material for the repair of connective tissues comprising: a) a cell
preparation enriched with mesenchymal stem cells, b) three-dimensional
extracellular matrix comprising a hyaluronic acid derivative. This
reference discloses the various ways to chemically treat hyaluronic acid
to alter its biophysical and biological properties. For instance, U.S.
Pat. No. 6,482,231 discloses treatment with formaldehyde or vinyl sulfone
to give rise to cross-linked gels. In addition, various hyaluronic acid
derivatives are disclosed, for example a partial or complete ester of
hyaluronic acid with an aliphatic, aromatic or araliphatic alcohol, and a
crosslinked hyaluronic acid derivative.

[0011]U.S. Pat. No. 6,596,274 discloses a biological material comprising
two components, wherein the first component comprises alternatively (1) a
culture of autologous or homologous bone marrow stem cells partially or
completely differentiated into specific connective tissue cellular lines
or (2) a sole extracellular matrix free from any cellular component
secreted by the specific connective tissue cellular lines; and a second
component comprises a three-dimensional biocompatible and biodegradable
matrix consisting of a hyaluronic acid ester having a degree of
esterification between 25 and 100%. The preferred hyaluronic acid ester
disclosed is the benzyl alcohol ester having a degree of esterification
varying from 25 to 100%.

[0012]U.S. Pat. No. 5,166,187 discloses a biomaterial consisting of an
association of collagen, chitosan acetylated to a degree of acetylation
between about 10% and about 40% and of glycosaminoglycans. The disclosed
biomaterial is used for making extracellular matrices for regeneration of
nerve cells and bones as well as biocompatible envelopes. A particular
application is the making of artificial skin consisting of a dermal
layer.

[0013]European Patent EP 1003567 B1 discloses a polysaccharide based gel
which comprises: (1) chitosan or a chitosan derivative; and (2) a salt of
polyol or sugar. The gel may be formed in situ within a tissue, organ or
cavities of an animal or human.

[0014]European Patent Application EP 0784985 A1 discloses a bioabsorbable
hydrophilic material comprising one or more compounds selected from a
group consisting of gelatin, collagen, a collagen derivative, chitosan, a
chitosan derivative, and triethanolamine alginate. A bone-forming graft
is also disclosed comprising a bone morphogenetic protein and the
bioabsorbable hydrophilic material.

[0015]European Patent Application EP 0544259 A1 discloses a water
insoluble biocompatible hyaluronic acid polyion complex that comprises
hyaluronic acid and at least one biocompatible high molecular compound
having amino or imino groups. The polyionic complex is made by reacting
an alkalimetal salt of hyaluronic acid with the high molecular compound
in an organic acid aqueous solution.

[0016]PCT patent WO 03/008007 A2 discloses an implantable device for
facilitating the healing of voids in bone, cartilage and soft tissue. The
device includes a cartilage region comprising a polyelectrolytic complex
joined with a subchondral bone region. Each of these regions comprise a
macrostructure of a bioresorbable polymer. The device also includes a
microstructure which is composed of various polysaccharides including
hyaluronic acid. The polyelectrolytic complex transforms to hydrogel,
following the implant procedure.

[0017]The present disclosure describes the use of a malleable cell culture
matrix comprising both hyaluronan and chitosan as well as a
polyelectrolytic complex (PEC) of the two constituents, that are combined
in their dry states prior to the formation of the polyelectrolyte
complex. The disclosure describes a new material that can function in
vitro as an malleable cell culture material for pluripotent cells and
subsequently perform as the delivery vehicle for implantation of these
same cells into a host tissue. The present application refers to
"hyaluronan" and "hyaluronic acid" as synonyms and both terms will
therefore be used interchangeably.

SUMMARY

[0018]One embodiment of the present invention is a composition comprising:
a hyaluronic acid viscoelastic gel, a chitosan hydrogel, and a network of
polyelectrolytic complex fibers comprised of chitosan and hyaluronic
acid. The compositions of the present invention may further comprise one
or more biologically active agents, such as drugs, cytotoxic agents,
pharmaceuticals, growth factor proteins, hormones, morphogens, phage
vectors, viri vectors, artificial chromosomes, antibiotics,
antineoplastics, and anticoagulants. In one embodiment, the biologically
active agent is paclitaxel, which may be in the form of a bioconjugate of
a low molecular weight hyaluronic acid and paclitaxel. The biologically
active agent may include a protein selected from of the transforming
growth factor-beta (TGF-β) family of proteins. The composition may
further comprise pluripotent cells, such as stromal cells, mesenchymal
stem cells, and mixtures thereof.

[0019]The chitosan hydrogel of the composition may be formed by adding an
aqueous solution to dry protonated chitosan. The hyaluronic acid
viscoelastic gel of the composition may be formed by adding an aqueous
solution to dry hyaluronic acid. A malleable three-dimensional cell
culture matrix and a tissue engineering material may be formed from the
composition.

[0020]Also provided is a composition comprising: a reaction product of
hydrated preprotonated chitosan and hydrated hyaluronic acid. Another
embodiment is a composition formed by the process of: forming a mixture
of dry hyaluronic acid and dry protonated chitosan, and adding an aqueous
solution to the mixture. Another embodiment is a device for the delivery
of tissue engineering materials comprising: a malleable cell composition
comprising a hyaluronic acid viscoelastic gel, a chitosan hydrogel, and a
network of polyelectrolytic complex fibers comprised of chitosan and
hyaluronic acid, and a polymer.

[0021]The polymers of the device may be a rigid biodegradable homopolymer
or a co-polymer comprising a biodegradable polymer, such as a
poly-(alpha-hydroxy acid) polymer or caprolactone. The device may be a
small diameter vascular graft. The polymer may be malleable
non-biodegradable homopolymer or a co-polymer. The device may include
biologically active agents and/or pluipotent cells.

[0022]Another embodiment is a method of forming a tissue engineering
composition comprising: forming a mixture of dry hyaluronic acid and dry
protonated chitosan, and adding an aqueous solution to the mixture,
wherein the aqueous solution forms a network of polyelectrolytic complex
fibers, and wherein the aqueous solution forms a hydrogel of homogeneous
chitosan and a viscoelastic gel of homogeneous hyaluronic acid. In the
method embodiments, the aqueous solution may be added dropwise. The
mixture may be comprised of mechanically blended dry hyaluronic acid and
dry protonated chitosan. The mixture may comprise about 0.25 to about
0.75 mole of hyaluronic acid to about 1 mole of chitosan and it may
include an effective amount of hyaluronic acid to produce a predetermined
amount of the network. The mixture may include an effective amount of
chitosan to produce a predetermined amount of the network. The aqueous
solution may be added in an amount of about 10:1 to about 15:1, based on
the weight of the mixture. Method steps may include adding one or more
biologically active agents and/or pluripotent cells.

[0023]Another embodiment is a method of delivering a composition into a
host tissue, comprising: forming a mixture of dry protonated chitosan and
dry hyaluronic acid, hydrating the mixture with an aqueous solution to
form a network of polyelectrolytic complex fibers, adding a biologically
active agent to the network, and delivering the network into a host
tissue.

[0024]Another embodiment is a method of growing tissue in vivo comprising:
injecting a tissue engineering material into mammalian host tissue,
wherein the tissue engineering material comprises pluripotent cells, a
hyaluronic acid component, a chitosan component, and a polyelectrolytic
complex component comprised of chitosan and hyaluronic acid, and allowing
the pluripotent cells to influence phenotypic choice of undifferentiated
cells resident within the host tissue.

[0025]One embodiment provides a malleable cell culture device comprising
unreacted regions of hyaluronic acid and chitosan invested with insoluble
fibers of a polyelectrolytic complex composed of hyaluronic acid and
chitosan. The device is capable of being metabolized by human stromal
cells, mesenchymal cells and their more differentiated decendents.
Another embodiment is a composition to serve as a medium for high
concentration, regional delivery of biologically active agents.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026]The present invention will be better understood, when taken in
conjunction with the following drawings, of which:

[0027]FIG. 1 represents a dry chitosan leaflet processed according to one
embodiment of the present invention.

[0028]FIG. 2 represents a dry blend mixture of hyaluronic acid and
chitosan according to one embodiment of the present invention.

[0029]FIG. 3 represents a dry blend mixture of hyaluronic acid and
chitosan according to one embodiment of the present invention.

[0030]FIG. 4 represents a dry blend mixture of hyaluronic acid and
chitosan according to one embodiment of the present invention.

[0031]FIGS. 5 and 6 show, at low magnification, the two reactants,
hyaluronic acid and chitosan, in their dry blended forms according to one
embodiment of the present invention.

[0032]FIG. 7 shows the reaction product after the hydration of the dry
hyaluronic acid and chitosan reactants according to one embodiment of the
present invention.

[0033]FIG. 8 shows a region of unreacted hyaluronic acid after
lyophilization according to one embodiment of the present invention.

[0034]FIG. 9 shows the HY-CT-PEC fiber network on the surface of an
unreacted chitosan leaflet after lyophilization according to one
embodiment of the present invention.

[0035]FIG. 10 represents viable cells after 5 days in culture, wherein a
three dimensional, malleable cell culture composition has been used as a
cell culture media according to one embodiment of the present invention.

DETAILED DESCRIPTION

[0036]In the following description, numerous specific details are set
forth in order to provide a thorough understanding of the invention. It
will be apparent however, to one of ordinary skill in the art, that the
invention may be practiced without limitation to these specific details.
In other instances, well known methods and structures have not been
described in detail so as not to unnecessarily obscure the invention.

[0037]It must be noted that as used herein and in the appended claims, the
singular forms "a", "an", and "the" include plural reference unless the
context clearly dictates otherwise. Thus, for example, reference to a
"cell" is a reference to one or more cells and equivalents thereof known
to those skilled in the art, and so forth. Unless defined otherwise, all
technical and scientific terms used herein have the same meanings as
commonly understood by one of ordinary skill in the art. Although any
methods and materials similar or equivalent to those described herein can
be used in the practice or testing of the present invention, the
preferred methods, devices, and materials are now described. All
publications mentioned herein are incorporated herein by reference, to
the extent the reference provides support for the present application.
Nothing herein is to be construed as an admission that the invention is
not entitled to antedate such disclosure by virtue of prior invention.

[0038]Devices and methods are disclosed herein for treating mammalian
bone, articular cartilage, cartilage of the nucleus pulposus and annulus
fibrosus, myocardium and various other connective deficiencies, defects,
voids and conformational discontinuities produced by congenital
deformities, osseous and/or soft tissue pathology, traumatic injuries,
and accidental, surgical, or functional atrophy. One device of the
present invention provides an initial microenvironment within which human
stromal cells, human mesenchymal cells and their differentiated progeny
may be cultured in vitro and subsequently delivered into a host tissue
defect and safely established therein within boundaries of invention.
Thus, the present invention provides the means and environment for
pluripotent cells to regenerate a specific tissue type, both in vitro and
in vivo, for example, as the device is capable of being transplanted into
connective tissue defects for the purpose of generating phenotypically
specific repair tissue.

[0039]Aspects of the present invention relate to methods of forming a
malleable hydrogel device with insoluble fibers or filaments therein, and
of forming a three dimensional, random and irregular network of
reinforcing fibers. The stiffness of the hydrogel component and density
of reinforcing fibers are controlled in one method of the present
invention to meet specific biologic demands of the cells to be grown
within the device and to meet particular surgical handling requirements.
The various compositions of the present invention are capable of being
metabolized by human stromal and mesenchymal cells as well as other cells
expressing the CD44 receptor.

[0040]One aspect of the present invention relates to a composition
comprising a dry protonated chitosan and a dry hyaluronic acid. FIGS. 2-6
represent a mixture of dry hyaluronic acid and chitosan of one embodiment
of the present invention. Another aspect of the present invention relates
to a method of forming dry chitosan leaflets. FIG. 1 represents dry
chitosan leaflets prepared by one method of the present invention.

[0041]Chitin is the major structural constituent of the exoskeleton of
crustaceans and insects and is a component of the cell wall of fungi.
Chitosan (poly-β1-4-glucosamine) is the highly deacetylated
form of chitin and is classified as an amino polysaccharide. Chitosan
(CT) is protonated by exposure to either mineral or organic acid and is
thus rendered soluble. Degree of CT protonation is related to solubility.
A minimum of about 45% protonation of the available amine groups renders
CT soluble. Preferably, chitosan is protonated from about 45% to about
100%. When exposed to pH levels below about 5.0, the amine groups
(--NH2) of chitosan molecules become protonated (--NH3.sup.+)
thus rendering the molecules soluble in water and providing them with a
strong positive charge (cation) that attracts negatively charged
molecules (anions). Protonation of between about 45% and about 100% of
available amine groups may be controlled to modify degree of interaction
with the anion. At certain degrees of protonation, chitosan may be
thought of as an amphoteric composition, meaning that it may both accept
and donate protons, although chitosan is traditionally thought of as a
cation in aqueous solution.

[0042]Protonation may occur by the exposure of chitosan to an acid to form
a solution, preferably the substantially stoichiometric addition of an
acid. The acid may be any inorganic or organic acid, preferably formic
acid or glacial acetic acid. After the chitosan is protonated by exposure
to an acid, the protonated CT solution may be lyophilized to a stiff
porous chitosan fabric. The architecture of this CT fabric may be that of
randomly sized, randomly shaped, intercommunicating interstices. CT
fabric dimensions and physical properties may be controlled by the
concentration of protonated CT in solution. The lyophilized CT fabric may
be reduced to individual leaflets or platelets of dry protonated
chitosan. These small pieces of CT fabric may have a thickness of about
1-10 micrometers and irregular planar shapes and dimensions. FIG. 1
represents a dry chitosan leaflet prepared according to one method of the
present invention.

[0043]Chitosan may be obtained with a wide range of molecular weights up
to weight average molecular weight of 1,000,000 or greater. In the
preferred compositions of this invention, CT in the Mw range of 600,000
to 900,000 Da may be employed. However, it is recognized that greater or
lesser Mw examples of CT may be employed to accommodate specific biologic
requirements of cells to be cultured within its hydrogel or meet specific
mechanical demands required of the final device.

[0044]Further, chitosan exhibits interesting biological properties that
may be used clinically. It is hemostatic and cicatrizing and may be used
as a cell culture support. Its antimicrobial capacity acts by stimulation
of the immune system and, in particular, it induces the activation of
macrophages.

[0045]Hyaluronic acid, also known as "hyaluronan," is a linear polyanionic
polysaccharide, and is a member of the family known as
glycosaminoglycans. It is present in most vertebrate connective tissues
at relatively high concentrations (up to 10 mg/ml). Hyaluronic acid (HY)
may be obtained with an original weight average molecular weight of about
1.8×106 Da. Upon size reduction to small particles, the Mw may
be reduced to 1.0×106 Da. As with its chitosan partner in this
construct, the molecular weight of HY may be altered to accommodate
specific biologic demands of cells to be cultured. The basic structural
unit of HY is a disaccharide consisting of D-glucuronic acid (GIcA) in
β1-3 linkage to N-acetyl-D-glucosamine (GIcNAc). The
disaccharides may be linked together in a β1-4 linkage. In its
highly hydrated form, hyaluronic acid shows unique properties of
viscoelasticity and plasticity.

[0046]In cartilage, hyaluronic acid plays a central role in the assembly
and maintenance of the macromolecular components constituting the
chondrocytes' extracellular matrix (ECM). It binds with high specificity
and affinity to aggregan and link protein. A single hyaluronic acid chain
may form a central "filament" that binds a large number of aggregan
molecules, forming a supermolecular complex that immobilizes water and
leads to a highly hydrated gel-like structure. In addition hyaluronic
acid binds with high affinity to the chondrocyte CD44 receptor.
Hyaluronic acid is naturally present in the vitreous humor of the eye and
in the synovial fluid of joint cavities. It is used in surgical
procedures involving the anterior chamber of the eye, such as corneal
transplants and the removal and replacement of a cataractous lens. It is
also used in the therapy of arthritis where injection of hyaluronic acid
into the joint space may restore the rheological properties of the
synovial fluid.

[0047]In its naturally occurring form hyaluronic acid is a salt, such as,
for example, a sodium salt. The naturally occurring form may be subject
to an ion exchange process to convert hyaluronic acid to an acid form.
Although the acid form is preferred, both forms of hyaluronic acid may be
used. Changing the naturally occurring form to an acid form may change
the traditionally anionic hyaluronic acid to a more amphoteric substance,
being able to both accept and donate protons in aqueous solution.

[0048]One embodiment of the present invention is a mixture of dry chitosan
and dry hyaluronic acid, as shown in FIGS. 2-6. Preferably, the dry
protonated chitosan is freeze-dried. The chitosan may be in the form of
individual flakes or leaflets, as seen in FIG. 1. The individual flakes
of chitosan comprise area dimensions of about up to 1 mm2 and
thickness dimensions of about 1-16 μm. The chitosan is preferably
protonated to the degree of about 45% to about 100% of available amine
groups.

[0049]Another aspect of the present invention relates to a composition
comprising a hyaluronic acid viscoelastic gel, a chitosan hydrogel and a
network of polyelectrolytic complex fibers surrounding and penetrating
both the HY viscoelastic gel and the CT hydrogel. Another aspect of the
present invention relates to a method of combining hyaluronic acid with
chitosan in their dry states and adding an aqueous solution to this dry
blend of reactants.

[0050]The mixtures of dry HY and dry CT may be hydrated by an aqueous
solution to form a reaction product. Once introduced to water, hyaluronic
acid forms a viscoelastic gel, while chitosan forms a hydrogel. The two
reactants form a polyelectrolytic complex at the regions of the intimate
physical contact. FIG. 7 represents a post-reaction composition according
to one embodiment of the invention. After reaction, there are regions of
a homogeneous viscoelastic gel and a homogeneous hydrogel, remaining
unreacted. Surrounding and penetrating these homogeneous regions of
viscoelastic gel and hydrogel is a three-dimensional network of insoluble
fibers comprising a polyelectrolytic complex of both CT and HY. See FIG.
7.

[0051]Polyelectrolytic complex (PEC) filaments may be formed from
glycosaminoglycans (GAG's) serving as the anion reacted with polycations
as well as other similarly structured compounds. While having the
requisite electron affinity for bonding, some of the sulfonated GAG's may
not be effective in supporting the appropriate cell-types. The PEC fibers
may be made from hyaluronic acid (HY), a non-sulfonated GAG, and chitosan
in one embodiment of the present invention.

[0052]While not wishing to be bound by theory, once introduced to an
aqueous media, the strong negative charge associated with HY (anion) may
be provided by the carboxylic acid group (-COO-H.sup.+) of its
glucuronic acid moiety. The amine groups (--NH2) of chitosan
molecules have been protonated (--NH3.sup.+), thus rendering the
molecules soluble in water and providing them with a strong positive
charge (cation) that attracts negatively charged molecules, such as
hyaluronic acid in solution, thus forming strong electrostatic
interactions between the two reactants. However, since both the degree of
protonation of the chitosan and the form of HY (acid or salt form) may be
varied, both compounds may be amphoteric, having the ability to both
accept and donate protons, in aqueous based solutions. Although HY is
traditionally thought of as the anion and chitosan is traditionally
thought of as the cation in aqueous solution, such strict labels may not
be appropriate as pH, degree of protonation, and the form of HY (salt or
acid based) may be varied and optimized.

[0053]When a solution of protonated chitosan is exposed to a solution of
HY, a polyelectrolytic complex (PEC) is formed. The reaction product of
hydrated CT and HY, according to embodiments of the present invention,
may be referred to as a PEC fiber reinforced hydrogel. Compounds
resulting from these strong electrostatic interactions are also known as
polyionic complexes (PIC). See FIG. 7.

[0054]In one embodiment of the invention, proportions for the reactants
and their physical sizes and locations relative to each other in the dry
state may be precisely controlled so that the homogeneous regions of HY
viscoelastic gel and CT hydrogel remain adjacent to one another while the
insoluble fibers of the PEC surround and penetrate both regions. In their
dry states, the leaflets of chitosan and the particles of hyaluronic acid
are thus mechanically blended in appropriate mass ratios. FIG. 5
represents the blended dry reactants, according to one embodiment of the
present invention.

[0055]For example, the dry CT and HY reactants may be mechanically blended
in the range of about 0.25 to about 0.75 moles of HY for every about 0.6
to about 1.0 moles of CT. The dry reactants may be blended together in a
high speed blender, for example. The lyophilized CT may optionally be
reduced to leaflets separately in the blender, prior to the addition of
dry HY to the blender. The blender may have blades that rotate at speeds
in excess of about 30,000 rpm.

[0056]In another embodiment, the dry anion and cation reactants are
hydrated with sterile water, sterile saline or other solution suitable
for human injection. These solutions may contain biologically active
agents such as drugs, cytotoxic agents, pharmaceuticals, growth factor
proteins, hormones, morphogens, phage vectors, viri vectors, artificial
chromosomes, antibiotics, antineoplastics, and anticoagulants. For
example, the water or saline may contain the pharmaceutical, paclitaxel,
conjugated with a low molecular weight hyaluronan. Though the resulting
PEC fiber reinforced hydrogel is devoid of cells, it may be implanted in
a host tissue void and function as a depot of biologically active agents
at superphysiologic concentrations capable of attracting host cells into
the construct and its vicinity and influencing their phenotypic choice
upon their entry into the device.

[0057]In another embodiment, the mixture of dry HY and CT particulates may
be exposed to nutrient media containing cells. The compositions and
devices of the present invention therefore may comprise pluirpotent
cells. The cell culture media may contain bone marrow stromal cells and
mesenchymal cells as well as influential growth factors, morphogens and
other biologically active agents. Upon exposure to the fluid of the
culture media, the mixture of dry HY and CT particles may enter solution
forming the PEC filaments while maintaining homogeneous regions of
viscoelastic HY gel, and CT hydrogel. Where these reactants are proximate
to one another, they react with each other to form the network of
reinforcing PEC fibers. Attendant cells may be trapped within boundaries
of the PEC fiber reinforced hydrogel. The entire hyaluronic
acid-chitosan-polyelectrolytic complex hydrogel (HCP-h) may be described
as a fluid mass, bounded by a defined network of thin membranes composed
of PEC fibers.

[0058]This complex of HCP-h microenvironment may be invested with cells
and their associated cell transfer solution and subsequently deposited
into a sterile cell culture supplied with an excess of cell culture
media. In this context the invention may function as a malleable, three
dimensional, in vitro, cell culture matrix. Subsequently, it may be
lifted en mass from its culture media and implanted into a host tissue as
an in vivo tissue engineering device. The HCP-h microenvironment may also
be a depot for high concentration regional delivery of drugs, growth
factors, morphogens and other biologically active agents.

[0059]The water, water solution containing biologically active components,
saline solution, cell culture media or cell transfer solution are
referred to hereinafter as "water" for simplicity of discussing the
hydration step of one embodiment of the present invention. The water may
be added to a small sample of the dry blended HY and CT components, for
example, in a drop-by-drop manner using a syringe. For example, about 10
μL to about 15 μL of water may be added for each 1 mg of dry
reactants. This drop-by-drop placement of water on the dry component
mixture may thus be controlled to create predetermined physical
properties of the final reaction product.

[0060]One embodiment of the present invention relates to compositions
comprising hyaluronic acid, chitosan, and the PEC fiber reinforced
hydrogel formed by these two components. These compositions can be used
as an extracellular matrix, a three-dimensional malleable cell culture
complex, an injectable tissue engineering material or a depot for
regional high concentration delivery of pluripotent cells, cytotoxic
agents and other biologically active agents. The physical shape, size and
mass ratio of the two reactant particles (in their dry states), as well
as their positions of proximity to one another, may be used to govern
chemical and physical properties of the resultant PEC fiber reinforced
hydrogel. Upon hydration with sterile water, saline, cell culture media
or cell transfer solution, the resulting complex contains multiple
homogeneous regions of unreacted hyaluronic acid and unreacted chitosan
surrounded by and penetrated by fibers of the polyelectrolytic complex.
FIGS. 8 and 9 are representations of the HY-CT-polyelectrolytic complex,
after lyophilization.

[0061]Another aspect of the present invention relates to a three
dimensional device for the delivery of tissue engineering materials.
Another aspect of the present invention relates to a method of forming a
composition for the delivery of tissue engineering materials. Another
aspect of the present invention relates to a method of implanting a
composition into a host tissue in order to influence phenotypic choice of
undifferentiated host (mesenchymal) cells. Another aspect of the present
invention relates to a method of repairing tissue. A further aspect of
the invention relates to a method of delivering cytotoxic agents and
other pharmaceuticals to a particular anatomic site at high
concentration.

[0062]Mesenchymal stem cells (MSC) are the formative pluripotential blast
cells found inter alia in association with capillaries (i.e., the
vascular pericyte), bone marrow, skeletal and smooth muscle, myocardium
and other connective tissues that are capable of differentiating into any
of the specific types of connective tissue cells such as adipocytes,
osteoblasts, chondrocytes, fibroblasts and myocytes (of smooth and
skeletal muscle as well as the myocardiocyte). In order to isolate human
MSC, it is necessary to isolate rare pluripotent mesenchymal stem cells
from other cells in the bone marrow or other MSC source. Bone marrow
cells may be obtained from the iliac crest, femur, tibia, spine, rib or
other medullary spaces. Other sources of human mesenchymal stem cells may
include embryonic yolk sac, placenta, umbilical cord, fetal and
adolescent skin, blood and other mesenchymal stem cell tissues.

[0063]Isolated human mesenchymal stem cell compositions serve as the
progenitors for various mesenchymal cell lineages. Isolated mesenchymal
stem cell populations have the ability to expand their numbers in culture
without differentiating and have the ability to differentiate into
specific phenotypic lineages when either induced in vitro or implanted in
vivo at the place of the damaged tissue.

[0064]A sample of bone marrow may be obtained from a patient by aspiration
from any one of several bone marrow deposits such as between cortical
plates of the ileum. Once harvested, adherent cells of marrow specimen,
consisting of bone marrow stromal cells (BMSC) and mesenchymal stem cells
(MSC), are separated from the non-adherent cell fraction consisting of
hematopoietic progenitor cells. The adherent cell moiety is culture
expanded to tens of millions of cells per milliliter. Following culture
expansion of adherent cell populations in two-dimensional culture
environments and release from their points of attachment, aliquots of the
cell/media suspension are withdrawn by a sterile, microliter pipette,
centrifuged into a concentrated cell pellet, resuspended into a
predetermined volume of cell transfer fluid (i.e., sterile saline or
other balanced salt solution) and deposited upon a specific mass of the
HCP-h material.

[0065]Prior to their introduction to the HCP-h material, it is recognized
that the h-BMSCs and h-MSCs can be exposed to various growth factors and
morphogens via the culture media, or transfected with various phage or
viri vectors (i.e. retrovirus, adenovirus or artificial chromosome), in
order to influence the phenotype selected by a preponderance of cells in
the population. Also, growth factors, morphogens and other biologically
active agents can be introduced into the cells' media prior to their
placement into the HCP-h material to further influence their phenotypic
choice and upregulate their endogenous production of the same growths
factors or morphogens. Similarly, after the cell complex is created,
comprising the HCP-h composition, it may be placed into an excess of cell
culture media that contains particular morphogens and other biologically
active agents that may diffuse into the cell complex and influence
phenotypic selection of cells contained therein.

[0066]Another aspect of the present invention relates to a composition
comprising a dry protonated chitosan, a dry high molecular weight moiety
of hyaluronic acid and a dry bioconjugate of very low molecular weight
hyaluronic acid and paclitaxel. Paclitaxel is a member of a class of
drugs known as taxanes, which have been commercialized as a cancer
medication under the trade name Taxol®. Paclitaxel has been known to
slow or stop the growth of cancer cells in vivo and is normally insoluble
in water. However, by conjugating paclitaxel with very low molecular
weight examples of hyaluronan (for example, HY oligossaccharides of Mw
5,000 to low Mw examples of hyaluronic acid up to 60,000) it becomes
soluble in water. Addition of lyophilized (dry) HY-paclitaxel
bioconjugate to the various compositions of the present invention, such
as the dry CT and dry HY composition, and further hydration produces a
malleable depot of cytotoxic agent that may be implanted into a tissue
defect created by excision of a malignant tumor or injected into a
malignant tumor mass. For example the three components of dry CT, dry HY
and the dry HY-paclitaxel bioconjugate could be hydrated according to a
method of the present invention.

[0067]In an alternative embodiment, the dry HY particles and dry chitosan
particles of one embodiment of the present invention may be hydrated with
a solution of the HY-Paclitaxel conjugate, having a viscosity about equal
to the viscosity of water, to form an HCP-h gel saturated with a
predetermined concentration of HY-paclitaxel conjugate. The addition of
the bioconjugate in solution results in the uniform distribution of the
drug throughout the HCP-h gel.

[0068]In either embodiment of the hyaluronan-paclitaxel, hyaluronic acid,
and chitosan reactions, there will be regions of unreacted HY, CT and
HY-paclitaxel in addition to the reaction product, PEC, in the resulting
post-reaction composition. The conjugate does not interact as an anion
with chitosan (cation) in the reaction. The conjugation of paclitaxel to
hyaluronan requires an attachment of intermediaries to hyaluronan's
carboxylic acid group (-COO-H.sup.+), thus eliminating availability
of the hyaluronan component of the conjugate as an anionic partner to CT
in the subsequent reaction. Thus, post-reaction, the HY-paciltaxel
bioconjugate remains a homogeneous, unreacted entity informally
throughout the HCP-h volume.

[0069]Another embodiment of the present invention is a three dimensional
device for the delivery of tissue engineering materials comprising the
HCP-h material, and a rigid biodegradable homopolymer or co-polymers of
rigid biodegradable polymers. Suitable homopolymers include those of the
poly(alpha-hydroxy acid) group or poly-C-caprolactone. Suitable
co-polymers of rigid biodegradable polymers include, for example, D,D-L,L
polylactic acid-co-glycolide.

[0070]Another embodiment of the present invention is a three dimensional
device for the delivery of tissue engineering materials comprising the
HCP-h material, and a malleable non-biodegradable homopolymer or
co-polymers of malleable non-biodegradable polymers. Suitable malleable
non-biodegradable polymers include, for example, various species of
polyurethanes.

[0071]The compositions according to the various embodiments of the present
invention may also contain additional components, such as morphogens that
are able to direct and enhance differentiation of mesenchymal stem cells
along a desired phenotypic lineage. Such bioactive factors or
combinations of factors are effective to induce differentiation of MSCs
in host tissue according to the present invention into a phenotypic
lineage selected from the group consisting of osteogenic, chondrogenic,
tendonogenic, ligamentogenic, myogenic, marrow stromagenic, adipogenic,
and dermatogenic.

[0072]In specific examples, the bioactive factor is a protein belonging to
the transforming growth factor-β super family (TGF-β), such as
TGF-β1, TGF-β3, or bone morphogenetic protein (BMP)
proteins. Other useful bioactive factors that could be delivered by the
present invention include, but are not limited to, platelet derived
growth factor (PDGF), insulin-like growth factor (IGF), and fibroblast
growth factor (FGF) and its several analogues.

[0073]Other therapies, including but not limited to drugs, biologically
active agents, and other agents, may also be utilized in or with the
HCP-h compositions; either to aid the function of the HCP-h or to cause
other stimuli. The drugs, biologics, or other agents may be naturally
derived or otherwise created (e.g. synthesized). For example, growth
factors can be derived from a living being (e.g. autologous, bovine
derived, etc.), produced synthetically, or made using recombinant
techniques (e.g., rh-BMP-2). Regardless of the time of investment or
incorporation of these materials, they may be in the form of solid
particulates, solution, gel or other deliverable form. Utilizing gel
carriers may allow for the materials to be contained after wetting, for
some tailorable length of time.

[0074]Malleable non-biodegradable polymers, including polyurethanes (PU)
or other materials used for development of small diameter vascular grafts
may also be used in an embodiment of the present invention. The HCP-h
substance may serve as a microstructure for these grafts, invested within
the interstices of polyurethane tubes possessed of 3-D architectures
within their walls. The architectures of the polyurethane grafts may be a
network of randomly sized, randomly shaped, infinitely intercommunicating
void spaces.

[0075]Polyurethane offers no physical resistance to the forces directing
microstructure solutions toward interior spaces of its 3-D architecture.
It is possible to saturate these interior void spaces with a blend of
hyaluronic acid and pre-protonated chitosan rendered as dry powders of
very fine particle size. The HY and CT powders may be added separately,
sequentially, or in a dry mixture blend. The grafting manufacturing
conditions preferable are sterile: For example, the following
manufacturing parameters might be used to invest the void spaces of a
polyurethane graft with the various HCP-h compositions of the present
invention: a class 100 clean room, laminar air flow, very low humidity
levels, very cool temps (i.e. 60-65° F.). Packaging the resulting
HCP-h invested polyurethane graft preferably includes: air tight glass
ampoules or blisters, inert package atmosphere (N2/argon). The HCP-h
compositions of various embodiments of the present invention therefore
may be invested as a microstructure within a malleable (polyurethane)
macrostructure tube.

[0076]A small diameter vascular graft is an example of the HPC-h material
and malleable polymer embodiment of the present invention. The preferred
embodiment for this device would a manufactured substitute for the
saphenous vein graft employed in coronary artery by-pass procedures.
Additional applications of the vein graft with the HPC-h material may act
as a microstructure are repair/regeneration of damaged urethra and
peripheral nerve regeneration.

DETAILED DESCRIPTION OF THE DRAWINGS.

[0077]FIGS. 1-4 detail dimensions of typical chitosan leaflets. FIG. 1 is
illustrative of a CT leaflet, processed according to one embodiment of
the present invention. The flat leaflet yield a good area measurement of
1 mm. FIGS. 2, 3 and 4 represent the hyaluronan (HY)/chitosan (CT) dry
blend mixtures of one embodiment of the present invention. These images
show the CT leaflets "on edge" for thickness measurements at
magnification. The chitosan leaflet has a thickness of 4.5 μm in FIGS.
2 and 3. FIG. 4 shows a chitosan leaflet with a 13 μm thickness. FIGS.
2-4 also show, at high magnification, the positional relationship of dry
CT leaflets and dry HY particles. FIGS. 5 and 6 show, at low
magnification, the general relationship of the two reactants in their dry
blended forms and provides visual cues to identify each reactant.

[0078]When these dry particles are simultaneously exposed to cell transfer
solution or culture media (i.e. water based solution), they both dissolve
in the water as they come in contact with it. Where CT and HY particles
are in intimate contact with each other in the dry state, an insoluble
polyelectrolytic complex (PEC) precipitates out of solution forming a
network of insoluble PEC fibers throughout the composition.

[0079]Where large regions of HY or CT are isolated from each other, there
will be collections of pure HY and CT solutions remaining after the PEC
reaction is completed. See FIG. 7. The dry blend of CT and HY therefore
is hydrated by solution first, the PEC forms in regions where the CT and
HY are proximate to one another, and ultimately there remain regions of
isolated un-reacted and substantially pure CT and HY in solution.
Physical placement of the reactants in their dry state is thus critical
to formation of the ultimate HY-CT polyelectrolytic fibers and can be
optimized accordingly. The reactant placement is critical with respect to
the dry CT leaflet as well. Where a large leaflet of CT is present and
physically removed from a significant mass of HY, the CT leaflet will
reform in solution post-reaction as a region of CT hydrogel.

[0080]FIG. 7 shows the HCP-h composition after the dry HY and CT reactants
have been hydrated and the PEC reaction completed. The resulting PEC
fiber reinforced hydrogel has also been lyophilized. Regions of unreacted
CT and HY are identified. FIG. 7 also shows the insoluble HY-CT-PEC
fibers. These leaflets of CT may have small HY particles on their
surfaces. At these positions, a HY-CT-PEC fiber will form on the
remaining surface of the CT leaflet. FIG. 8 shows a region of unreacted
HY after lyophilization. FIG. 9 shows the fiber network on the surface of
an unreacted CT leaflet after lyophilization.

[0081]FIG. 10 illustrates viable cells after 5 days in a culture
comprising the composition of a network of HY-CT-PEC fibers. HT-1080
fibrosarcoma cells have been grown in the culture. The cells have been
injected with green florescent protein in enhance visibility.

[0082]The HY-CT-PEC fibers remain insoluble and made be thought of as the
insoluble particles suspended in a fluid medium, such as the blend of
insoluble and soluble components of a colloid. Unreacted HY and CT
materials are soluble and together form the viscoelastic and hydrogel
components of one composition embodiment of the present invention. The
net result of adding water to the dry blend of HY and CT particles is a
fiber (PEC) reinforced mixture of viscoelastic gel (HY) and hydrogel
(CT).

[0083]Thus since the invention disclosed herein may be embodied in other
specific forms without departing from the spirit or general
characteristics thereof, some of which forms have been indicated, the
embodiments described herein are to be considered in all respects
illustrative and not restrictive. The scope of the invention is to be
indicated by the appended claims, rather than by the foregoing
description, and all changes which come within the meaning and range of
equivalency of the claims are intended to be embraced therein.